Investigations on the oligosaccharide units of an A myeloma globulin

The carbohydrate content of an A myeloma globulin was investigated. The carbohydrate content was found to be unchanged when the protein was isolated from the patient over a period of 18 months. The various polymeric forms of the protein contained similar proportions of carbohydrate. The A myeloma globulin contained approx. 2 residues of 6-deoxy-l-galactose (l-fucose), 14-15 of d-mannose, 12-13 of d-galactose, 12-13 of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine), 6 of 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine) and 5 of N-acetylneuraminic acid (sialic acid), and these were distributed between six oligosaccharide units all of which were present on the heavy polypeptide chains. The oligosaccharide units showed two kinds of heterogeneity, which have been termed central and peripheral. Central heterogeneity was shown by the presence of three completely different core units, which had the following compositions: (1) 3 residues of d-galactose and 3 of 2-acetamido-2-deoxy-d-galactose, joined to protein by an O-glycosidic linkage between acetamidohexose and serine; (2) 3 residues of d-mannose, 2 of d-galactose and 3 of 2-acetamido-2-deoxy-d-glucose, joined to protein by an N-glycosidic linkage between acetamidohexose and aspartic acid; (3) 4 residues of d-mannose and 3 of 2-acetamido-2-deoxy-d-glucose with a linkage similar to that in (2). The core oligosaccharide units showed peripheral heterogeneity in the attachment of 6-deoxy-l-galactose, 2-acetamido-2-deoxy-d-glucose and N-acetylneuraminic acid. Tentative structures are proposed for these various types of oligosaccharide unit. Glycopeptides were isolated in which the sialic acid content exceeded that of d-galactose. Explanations are given for the electrophoretic mobility and staining characteristics of the various glycopeptides.     

Glycopeptides of immunoglobulins: Investigations on IgA myeloma globulins

The oligosaccharide units of a type K and a type L IgA immunoglobulin have been examined. The two proteins differed in their content of 6-deoxy-l-galactose and N-acetylneuraminic acid, and in the d-mannose/d-galactose ratio. With glycopeptides prepared from the type K protein, specific glycosidases liberated the N-acetylneuraminic acid and 7-8 residues of 2-acetamido-2-deoxy-d-glucose, and mild acid hydrolysis released most of the 6-deoxy-l-galactose. The type K immunoglobulin appeared to contain 3 oligosaccharide units, whereas the type L protein probably contained 3 or more units.     

A study of the carbohydrate present in three type K macroglobulins

For a monomeric molecular weight of 180000 three type K macroglobulins (IgM) contained 6-deoxygalactose, mannose, galactose, 2-acetamido-2-deoxyglucose and N-acetylneuraminic acid in the molar proportions 5:38:11:27:7 for Row IgM, 5:31:9:21:7 for Sha IgM, and 5:29:11:26:8 for Tya IgM. The first two proteins were euglobulins whereas Tya IgM was a pseudoglobulin, and therefore the total content of carbohydrate does not appear to be related to the physicochemical properties of the proteins. The three proteins appeared to contain different numbers of oligosaccharide units, Row IgM having about ten units/monomer, and Sha IgM and Tya IgM about eight each. All three proteins had two types of oligosaccharide unit, which by analogy with an immunoglobulin A myeloma globulin were called Type 2 and Type 3 respectively. The Type 2 units had molecular weights equal to or greater than 2000 and contained 1 residue of 6-deoxygalactose, 3-4 of mannose, 1-2 of galactose, 3-4 of 2-acetamido-2-deoxyglucose and 0-2 of N-acetylneuraminic acid. The Type 3 units had molecular weights of less than 2000 and contained 0-1 residue of 6-deoxygalactose, 3-6 of mannose, 0-1 of galactose, 1-3 of 2-acetamido-2-deoxyglucose and no N-acetylneuraminic acid. Glycopeptides corresponding to the two types of unit varied in their aspartic acid content in that most of the Type 3 glycopeptides possessed only 1 residue of aspartic acid whereas most of the Type 2 glycopeptides had an average content greater than 1 residue.     

Structure of carbohydrate chains of a high-molecular-weight pulmonary glycoprotein

A human, alveolar glycoprotein having an apparent mol. wt. of 250 000 gave two major glycopeptide fractions (I and II) by Pronase digestion, followed by gel filtration, DEAE-cellulose column chromatography, paper chromatography, and paper electrophoresis. Glycopeptide I contained d-galactose, d-mannose, 2-acetamido-2-deoxy-d-glucose, and N-acetylneuraminic acid in the molar ratio of 2:3:4:1, whereas these sugars were present in Glycopeptide II in the molar ratio of 2:3:4:2.l-Fucose was present only in Glycopeptide II at a concentration of one l-fucose per three d-mannose residues. In both glycopeptides, 2-acetamido-2-deoxy-d-glucose was linked to an asparagine residue of the peptide chain. Based on the results of alkaline borohydride treatment, periodate oxidation, methlylation analysis, and sequential glycosidase degradation of the glycopeptides, tentative structures are proposed for both glycopeptides.     

13C-n.m.r.-spectral study of galactosyltransferase specificity with regard to the carbohydrate side-chain of native ovalbumin

As a prelude to studies using bovine N-acetylglucosaminide-β-(1→4)-galactosyltransferase to label membrane-surface glycoproteins with isotopically enriched d-galactose, the structural specificity of the enzymic reaction with water-soluble, hen ovalbumin has been examined. The enzyme-catalyzed transfer of d-galactose from UDP-d-galactose requires a (nonreducing) terminal 2-acetamido-2-deoxy-d-glucosyl group and exhibits selectivity towards saccharide chains containing d-mannose. This study considers the structural specificity of the enzyme with regard to the anomeric linkage between 2-acetamido-2-deoxy-d-glucose and d-mannose in the carbohydrate chains of hen ovalbumin. Uniformly ¹³C-enriched d-galactose was enzymically attached to the ovalbumin carbohydrate chain (which exhibits microheterogeneity in its structure), the protein was hydrolyzed, and separate glycopeptide fractions were chromatographically isolated. The ¹³C-n.m.r. spectra (60.5 MHz) of the fractions revealed two peaks for the anomeric carbon atom of d-galactose. The two peaks, at 104.20 and 104.39 p.p.m., were ascribed to d-galactosyl groups attached to 2-acetamido-2-deoxy-d-glucose respectively linked β-(1→4) and β-(1→2), to d-mannose in the glycopeptide chains. Quantifying of the spectral data revealed no specificity of d-galactosyltransferase towards the linkage from the terminal 2-acetamido-2-deoxy-d-glucosyl group to the penultimate d-mannosyl residue.     

A mouse submandibular sialomucin containing both N- and O-glycosylic linkages

A sialomucin from mouse submandibular glands was treated with mild base-Me₂SO. This treatment cleaves O-glycosylically linked oligosaccharides, but preserves the integrity of the protein core. After treatment with mild base-Me₂SO, 49.2% (by weight) of the oligosaccharides were removed from the polypeptide; they were composed of residues of 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-galactose, sialic acid, and d-galactose. These oligosaccharides were linked O-glycosylically via 2-acetamido-2-deoxy-d-galactose. Chromatography of the base-Me₂-SO-treated mucin on Sephacryl S-300 indicated that the protein core, with its base-resistant oligosaccharides, is a single, high-molecular-weight species. The mild-base-resistant linkages remaining on the protein core (50.8% of the total carbohydrates by weight) also contained d-mannose. The presence of these mild-base-resistant linkages, and the formation of 2-acetamido-2-deoxy-d-glucitol following treatment with m NaOH-m NaBH₄, confirmed the presence of N-glycosylic linkages.     

Preparation of anomeric pairs of 1-thioglycosides: use of anomerization catalyzed by boron trifluoride

Anomeric pairs of some alkyl 1-thioaldopyranosides of d-galactose, d-glucose, d-mannose, 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-galactose, and l-fucose were prepared. The per-O-acetylated, 1,2-trans anomers of 6-(trifluoroacetamido)hexyl 1-thioaldopyranosides and 5-(methoxycarbonyl)pentyl 1-thioaldopyranosides were anomerized with boron trifluoride in dichloromethane. The anomeric mixtures were then separated by chromatography, using columns of either silica gel or an ion-exchange resin. De-blocking of the separated compounds provided pure anomers of 6-aminobexyl 1-thioaldopyranosides or 5-carboxypentyl 1-thioaldopyranosides. The aglycons of the latter glycosides were further extended by reaction with aminoacetaldehyde diethyl acetal, which, after deacetalization of the products, provided an ω-aldehydo group. These series of glycosides could be readily coupled to proteins or solid matrices.     

Structure of O-specific side chains of lipopolysaccharides from Yersinia pseudotuberculosis

Lipopolysaccharide prepared from cells of Yersinia (Pasteurella) pseudotuberculosis of serogroups I, II, III, IV, and V is known to contain the 3,6-dideoxyhexose (DDH) paratose, abequose, paratose, tyvelose, and ascarylose in its respective O-specific side chains. Lipopolysaccharides or lipid-free polysaccharides of all of the 10 known serogroups and subgroups were subjected to methylation analysis and determined as alditol acetates by gas-liquid chromatography and mass spectrometry. The results indicated that the O-specific side chains of nine serotypes are composed of oligosaccharide repeating units in the form of four alternative general structures in which a terminal DDH may vary. These structures are DDH [Formula: see text] 6-deoxy-d-manno-heptose [Formula: see text] d-galactose (serogroups IA, IIA, and IVB), DDH [Formula: see text] d-mannose [Formula: see text] l-fucose (serogroups IB and IIB), and two configurations similar to the latter except that the 4-position of l-fucose was either linked to the d-mannose residue (serogroups VA and VB) or to the DDH residue (serogroups III and IVA). In contrast, O-groups in lipopolysaccharide of the newly discovered serogroup VI contained the DDH colitose and 2-acetamido-2-deoxy-d-galactose. Accordingly, all five known types of DDH have now been detected in lipopolysaccharides of Y. pseudotuberculosis. The sugar 6-deoxy-d-manno-heptose, present in O-specific side chains of serogroups IA, IIA, and IVB, has not yet been reported to occur elsewhere in nature.     

Thermal decomposition of β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-hexopyranoses under neutral conditions

β-d-Galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-glucose (LNB) and β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-galactose (GNB) decompose rapidly upon heating into d-galactose and mono-dehydrated derivatives of the corresponding 2-acetamido-2-deoxy-d-hexoses, including 2-acetamido-2,3-dideoxy-hex-2-enofuranoses and bicyclic 2-acetamido-3,6-anhydro-2-deoxy-hexofuranoses. The decomposition is conducted under neutral conditions where glycosyl linkages are generally believed to be stable. The half-lives of LNB and GNB were 8.1 min and 20 min, respectively, at 90 °C and pH 7.5. The pH dependency of decomposition rates suggests that the instabilities are an extension of the conditions for the peeling reaction, often observed with glycans of O-linked glycoproteins under alkaline conditions. Such decomposition under the neutral conditions is commonly observed with 3-O-linked reducing aldoses.     

Immunochemical studies on the specificity of soybean agglutinin

The specificity of the purified soybean agglutinin has been studied immunochemically by quantitative precipitin and quantitative precipitin inhibition assays. The lectin is precipitated by human A and Le^a blood-group substance, by the products of the second, third, fourth, and fifth stages of periodate oxidation of a human H blood-group substance (JS), and by precursor blood-group substances, as well as by a pig-submaxillary mucin having blood-group A activity, by partially hydrolyzed blood-group B substances (Pl fraction), and by group C streptococcal polysaccharide. The activity is attributable to terminal α-linked 2-acetamido-2-deoxy-d-galactopyranosyl or to α- or β-d-galactopyranosyl residues. The lectin did not precipitate with human blood-group H substances, with the product of the first stage of periodate oxidation (JS), with streptococcal group A polysaccharide, or with pig-submaxillary mucin devoid of blood-group A activity, and is poorly precipitated by blood-group B substances. Inhibition of precipitation with various monosaccharides indicated that the lectin is strongly specific for 2-acetamido-2-deoxy-d-galactose and for its oligosaccharides, and to a lesser extent for d-galactose and its oligosaccharides; the α-glycosides of both sugars were slightly more reactive than the β-glycosides of 2-acetamido-2-deoxy-d-galactose, and both α- and β-glycosides were more active than the free monosaccharides. Aromatic α- and β-glycosides of 2-acetamido-2-deoxy-d-galactose and d-galactose were better inhibitors than the corresponding methyl or ethyl compounds. The blood-group A trisaccharide α-d-GalNAcp-(1→3)-β-d-Galp-(1→3)-d-GlcNAc was more active than the disaccharide lectins by the use of precipitation with polysaccharides, as well as inhibition reactions, is essential to the understanding of their reactivity with cell-surface receptors.     

A specific method for d-galactose quantitative determination: A modification of the d-galactose oxidase assay

After treatment with d-galactose oxidase to form an aldehyde group, d-galactose or 2-acetamido-2-deoxy-d-galactose reacted with indole-hydrochloric acid to give a colored compound having a spectrum very similar to that of d-galacturonic acid, but with a maximum at 500 nm and a shoulder at 480 nm. The reaction is linear between 16.6 and 83 nmol of sugar per mL of final solution. 2-Amino-2-deoxy-d-galactose gave no reaction, even when 5 μmol were used, and 2-deoxy-d-lyxo-hexose did not interfere either.     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin.

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

The structure of a sulfated glycoprotein of chick allantoic fluid: methylation and periodate oxidation.

The structure of an antigenic, sulfated glycoprotein from chick chorioallantoic fluid has been investigated by exogalactosidase digestion, methylation and mass spectral analyses, periodate oxidation, and Smith degradation. The main carbohydrate chains are composed of D-galactosyl residues linked at C-3 and 2-acetamido-2-deoxyglucose residues linked at C-4. Fucose and N-acetylneuraminic acid residues are nonreducing terminal groups, and the N-acetylneuraminic acid groups are linked to the D-galactose residues at C-3. Most of the sulfate groups (91% of the sulfate) are located on C-6 of the 2-acetamido-2-deoxyglucose residues, and the rest on C-6 of the D-galactose residues. A large number of the D-galactose residues (36.9% of the total) are present as nonreducing terminal groups and another 21.7% of the D-galactose residues are in penultimate position to the nonreducing terminal N-acetylneuraminic acid residues. Although mild periodate oxidation indicates the presence of D-galactose in furanoside form (5.5% of total D-galactose), no 5-O-methyl derivative of D-galactose was observed on methylation.     

The structure of the polysaccharide O-chain of the LPS from Acinetobacter baumannii strain ATCC 17961

The gram-negative bacterium Acinetobacter baumannii strain ATCC17961 has been used by several laboratories in mouse models of respiratory A. baumannii infection, and a study of the role of its lipopolysaccharide in the pathogenicity is of interest. The structure of the O-deacylated polysaccharide O-chain component of its LPS has been determined by 2D NMR spectroscopy and mass spectrometry methods, and by the structural identification of oligosaccharides obtained by sequential application of the Smith degradation of the O-antigen. The O-chain was determined to be a polymer of a branched pentasaccharide repeating unit composed of 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid, 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-galactose, d-glucose, and d-galactose, and has the following structure:     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

The structure of unit B-type glycopeptides from porcine thyroglobulin

The structure of Unit B-type glycopeptides (monosialo-type and disialo-type) was investigated by Smith degradation, methyllation, and mass spectral analysis. These glycopeptides contain three peripheral sugar chains. Two are composed of D-galactose residues linked at C-6 and 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and the other is composed of a D-galactose residues linked at C-6, a 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and a D-mannose residue linked at C-2. Most of these peripheral sugar chains are linked to two inner D-mannose residues which are substituted at C-3 and C-6, and constitute branching points. L-Fucose and N-acetyl-neuraminic acid residues are nonreducing terminal groups, and a di-N-acetylchitobiose moiety is linked to an asparagine residue in the peptide moiety. By methylation analysis of the oligosaccharide obtained by hydrazinolysis of the disialoglycopeptide, the L-fucose residues was found to be linked to C-6 of the 2-acetamido-2-deoxy-D-glucose residue linked to the asparagine residue. From these results, and from the previously reported data on the sugar sequence and the anomeric configurations of the linkages between sugar residues, structures for these glycopeptides are proposed.     

Photochemical addition of 2-propanol and of acetone to methyl 5-deoxy-2,3-O-isopropylidene-β-d-erythro-pent-4-enofuranoside

High-capacity adsorbents for lectins, including Lotus tetragonolobusl-fucose-binding protein, were readily prepared by conjugation of monosaccharides with commercially available, epoxy-activated Sepharose. Purified, radioiodinated lectins were bound to cells of the mosquito Aedes aegyptii and of human KB tumour. Relative to human KB cells, mosquito cells bound less of lectins specific for the sugars (l-fucose and d-galactose) that are terminal residues in many mammalian glycoproteins, whereas the number of binding sites of lectins specific for core-region sugars (d-mannose and 2-acetamido-2-deoxy-d-glucose) were similar. Neuraminidase, which greatly enhanced binding of peanut agglutinin or soybean agglutinin to human KB cells, had negligible effects on binding of these lectins to mosquito cells. The comparative structures of surface oligosaccharides of mosquito and KB cells are discussed in relation to the lectin-binding studies.     

The chemical structure of a fragment of Micrococcus lysodeikticus cell-wall.

A fragment of Micrococcus lysodeikticus cell-wall obtained by cetylpyridinium recipitation from the nondialyzable portion of the degradation products of egg-white lysozyme was studied by the periodate oxidation and methylation procedures. The fragment consists of a polysaccharide chain composed of about 40 repeating (1 leads to 4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranosyluronic acid)-(1 leads to 6)-O-(alpha-D-glucopyranosyl) residues with D-glucopyranosyl residues at both ends. The alpha-D-glucopyranose residue at the reducing end is linked to a phosphate group that is also linked to C-6 of a 2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl residue of a peptidoglycan chain composed of four repeating (1 leads to 4)-O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl] residues. The peptidoglycan chain has, as nonreducing group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, and, as reducing residue, a 2-acetamido-3-O-(D-1-carboxytheyl)-2-deoxy-beta-D-glucose residue.